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Protein crystallization is a critical method for investigating protein structure and function, and it plays a vital role in understanding the mechanisms of biomacromolecular interactions, disease pathogenesis, and drug development. It involves the process of protein molecules stacking regularly in a solution to form ordered crystals, which is influenced by multiple factors such as protein purity, concentration, and buffer composition. To obtain high-quality crystals, steps including sample preparation, condition screening, and optimization are required. Research on protein crystallization drives advancements in the fields of biology and medicine, and provides support for drug design and protein engineering. This article outlines the key points of protein crystallization, including preparation, methods, screening, and optimization, to serve as a reference for related research.
1. The History of Protein Crystallization Development
Research on protein crystallization began in the mid-19th century, when Hunefeld first reported hemoglobin crystals. In the 20th century, crystallization technology became an important method for protein purification and research—notably, John Jacob Abel’s crystallization of insulin and James B. Sumner’s confirmation that enzymes are crystalline proteins. In the late 1930s, X-ray analysis was first applied to protein crystals, opening a new chapter in structural biology. With the development of genetics and molecular biology, especially the application of DNA technology, research on protein crystallization has undergone significant acceleration and transformation since the 1980s.
2. Protein Crystallization: A Combination of Art and Science
Despite the convenience brought by advancements in modern molecular biology and crystallography for many experiments, protein crystallization remains a challenge that is both simple and complex. This process combines scientific methods with artistic intuition and creativity, while also being highly dependent on the accumulation of experience.
Since biomacromolecules (e.g., proteins) are composed of a large number of different atoms arranged in complex ways, the crystallization process involves numerous variables and influencing factors. These factors include protein purity, concentration, pH value, and ionic strength—each of which can have a significant impact on crystallization results (Tables 1 and 2).
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Table 1. Chemical Variables Affecting Protein Crystal Growth |
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Purity of the sample |
Genetic modification |
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Conformational flexibility of the sample |
Molecular symmetry |
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Homogeneity of the sample |
Stability and denaturation degree of the sample |
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pH value and buffer |
Isoelectric point |
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Type and concentration of precipitant |
Presence or absence of His-tags and other purification tags |
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Concentration of the sample |
Thermal stability |
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Additives, cofactors, ligands, inhibitors, effectors, and excipients |
pH stability |
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Chaotropes (e.g., urea, guanidine hydrochloride, ammonium sulfate) |
Historical processes of sample acquisition, handling, and storage |
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Detergents |
Proteolysis |
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Metal ions |
Microbial contamination |
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Ionic strength |
Sample preservation |
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Reducing agents or oxidizing agents |
Sample handling and associated cleanliness |
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Source of the sample |
Types and concentrations of anions and cations |
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Amorphous particulate matter |
Relative supersaturation |
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Post-translational modifications |
Initial and final concentrations of other reagents |
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Chemical modifications |
Equilibration path and rate |
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Table 2. Physical Variables That May or Do Affect Protein Crystal Growth |
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Temperature |
Electromagnetic field |
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Equilibration rate |
Surface of the crystallization device |
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Crystallization method |
Viscosity of reagents |
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Gravity, convection, and sedimentation |
Heterogeneous and epitaxial nucleants |
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Vibration and sound |
Geometry of the crystallization device |
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Volumes of sample and reagents |
Time |
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Pressure |
Dielectric properties of reagents |
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Crystallization experiments typically start with screening, which aims to identify the key variable combinations that can produce crystals. However, to obtain crystals with specific properties (e.g., for structural biology, purification, or biotherapeutic research), further optimization experiments are often required (Tables 3 and 4).
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Table 3. Crystallization Methods – Achievement of Supersaturation |
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Vapor Diffusion Method (Sitting, Hanging, Sandwich) |
Sequential Extraction (a data processing and analysis technique) |
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Batch Crystallization Method (Microbatch Crystallization) |
pH Induction (simple, controllable) |
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Dialysis Technique (Microdialysis Technique plays an auxiliary role) |
Temperature Induction (easy to operate, low cost; needs to be used in combination with other methods) |
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Free Interface Diffusion (Counter-Diffusion, Liquid Bridge) |
Effector Addition (also referred to as "Silver Bullet") |
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Controlled Evaporation |
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Table 4. Reagents Used in Protein Crystallization |
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Salts (Ammonium Sulfate, Sodium Formate, Ammonium Phosphate, etc.) |
Non-Volatile Organic Compounds ((±)-2-Methyl-2,4-Pentanediol, 1,6-Hexanediol, Glycerol, etc.) |
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Polymers (Polyethylene Glycol [molecular weight range: 200–20,000], Ethylene Imine, Jeffamine®, etc.) |
Buffers (HEPES, Tris, Sodium Acetate, MES, etc.) |
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Volatile Organic Compounds (2-Propanol, 1,4-Dioxane, Ethanol, etc.) |
Additives (Calcium Chloride, Sodium Chloride, Tris(2-carboxyethyl)phosphine, n-Octyl-β-D-glucoside, etc.) |
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Although modern technology provides abundant resources and tools, crystallization is not merely a matter of using these tools. A successful crystallization strategy needs to be based on certain fundamental principles. As proposed by pioneers in the field of crystallization such as Alex McPherson, considering these principles before conducting experiments will lay a solid foundation for your crystallization work.
3. Key Elements of Protein Crystallization
· Sample: The purity of the sample is the cornerstone of successful crystallization, and careful handling is required to ensure high homogeneity of the sample.
· Homogeneity: The consistency of the chemical structure and molecular weight of the protein sample is crucial for the formation of ordered crystals.
· Solubility: Optimize the dissolution conditions of the sample in the buffer, pursue monodispersity, and avoid aggregation and precipitation.
· Stability: Ensure that the protein maintains structural and functional stability during crystallization and prevent denaturation.
· Supersaturation: Induce the ordered arrangement of protein molecules to form crystal nuclei by controlling supersaturation.
· Association: Regulate the ordered interactions between protein molecules to promote ordered aggregation and avoid non-specific aggregation.
· Nucleation: Control the formation of initial crystallization nuclei of proteins in the solution to optimize the number, size, and quality of crystals.
· Diversity: Try a variety of methods and conditions to identify suitable crystallization conditions for specific proteins.
· Control: Precisely regulate experimental conditions and processes to ensure the accuracy and reproducibility of experimental results.
· Impurities: Maintain sample purity and reduce the interference of impurities on crystallization and subsequent research.
· Preservation: Properly protect the crystallized protein crystals to prevent damage and ensure the reliability of subsequent research.
Hampton Research (USA), whose products are distributed by Beijing XMJ Technology Co., Ltd., is a leading manufacturer specializing in protein crystallization research. Its extensive product portfolio covers a full range of products, from initial crystallization screening and crystallization optimization screening to custom single-component crystallization reagents and crystallization growth optimization reagents. These products can meet the needs of different protein crystallization research, including the crystallization of general proteins, membrane proteins, and protein complexes.
With its cutting-edge biomacromolecular crystallization technology and practical, diverse product line, Hampton Research (distributed by XMJ) provides comprehensive crystallization research reagents and laboratory consumables to crystal researchers worldwide. These products not only greatly improve the work efficiency of researchers but also help them achieve outstanding scientific research results. Hampton Research has become one of the most trusted brands in the field of crystallization research.
Best-Selling Protein Crystallization Products of Hampton Research (Distributed by XMJ)
Hampton Research Protein Crystallization Screening Kits
In addition to the aforementioned products, XMJ also provides a variety of crystallization condition screening kits, including GRAS Screen?, the Grid Screen series, Low Ionic Strength Screen, XP Screen, JBScreen Wizard, and more. For inquiries and orders, please feel free to send email to info@xmjsci.com
Hampton Research Protein Crystallization Optimization Kit.
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Brand |
Item Number |
Product Name |
Specification |
Introduction |
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Hampton |
HR2-072 |
Solubility & Stability Screen |
0.5 ml, Deep Well block format |
Solubility and Stability Screening, Including 96 Conditions |
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HR2-413 |
Solubility & Stability Screen 2 |
0.5 ml, Deep Well block format |
Solubility and Stability Screening, Including 96 Conditions |
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Hampton |
HR2-070 |
Slice pH |
0.5 ml, Deep Well block format |
pH Screening Kit, Including 96 Conditions |
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Hampton |
HR2-138 |
Additive Screen |
1 ml, Deep Well block format |
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Hampton |
HR2-459 |
GRAS Additive |
1 ml, Deep Well block format |
GRAS Additive Screening, Including 96 Conditions
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Hampton |
HR2-096 |
Silver Bullets |
0.50 ml, Deep Well block format |
Additive Screening Kit, Including 96 Conditions
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Hampton |
HR2-407 |
Detergent Screen |
0.25 ml, Deep Well block format |
Additive Screening Kit, Including 96 Conditions
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Hampton |
HR2-214 |
Ionic Liquid Screen |
0.5 ml, tube format |
Ionic Liquid Screening Kit, Including 24 Conditions
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Hampton |
HR2-241 |
StockOptions pH |
10 ml, tube format |
Crystal Growth Screening Kit, Including 45 Conditions
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Hampton |
HR2-235 |
StockOptions Sodium Citrate |
10 ml, tube format |
Crystal Growth Screening Kit, Including 24 Conditions
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Hampton |
HR2-239 |
StockOptions Sodium Cacodylate |
10 ml, tube format |
Crystal Growth Screening Kit, Including 24 Conditions
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Hampton |
HR2-243 |
StockOptions MES |
10 ml, tube format |
Crystal Growth Screening Kit, Including 20 Conditions
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Hampton |
HR2-103 |
StockOptions Bis-Tris Propane |
10 ml, tube format |
Crystal Growth Screening Kit, Including 33 Conditions
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Hampton |
HR2-102 |
StockOptions HEPES |
10 ml, tube format |
Crystal Growth Screening Kit, Including 15 Conditions
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Hampton |
HR2-227 |
StockOptions Polymer |
10 ml, tube format |
Crystal Growth Screening Kit, Including 23 Conditions
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Hampton |
HR2-245 |
StockOptions Salt |
10 ml, tube format |
Crystal Growth Screening Kit, Including 49 Conditions
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HR2-073 |
CryoPro |
1 ml, tube format |
Crystal Cryoprotectant Screening, Including 48 Conditions |
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Hampton |
HR2-501 |
50% v/v Jeffamine® M-600® pH 7.0 |
200 ml |
Crystalline grade Jeffamine M-600 is used for the optimization of crystallization conditions. |
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Hampton |
HR2-525 |
50% w/v Polyethylene glycol 1,500 |
200 mL |
Crystalline grade PEG 1500 is used for the optimization of crystallization conditions. |
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Hampton |
HR2-527 |
50% w/v Polyethylene glycol 3,350 Monodisperse |
200 mL |
Crystalline grade PEG 3350 is used for the optimization of crystallization conditions. |
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Hampton |
HR2-529 |
50% w/v Polyethylene glycol 4,000 |
200 mL |
Crystalline grade PEG 4000 is used for the optimization of crystallization conditions. |
The single-component conditions included in the aforementioned kits can be ordered individually. In addition to the kits mentioned above, Ximeijie also provides a variety of crystallization optimization screening kits and reagents, including Proti-Ace?, Silica Hydrogel Kit, Heavy Atom Screens, I3C Phasing Kit, etc. For inquiries and orders, please feel free to send email to info@xmjsci.com.
Hampton Research Protein Crystallization Consumables and Tools
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Brand |
Item Number |
Product Name |
Specification |
Introduction |
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HR2-320 |
Seed Bead Kit |
24 tubes with PTFE Seed Bead |
Seed Preparation Beads |
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Hampton |
HR3-105 |
MASTERBLOCK® 96 Deep Well polypropylene plate |
50 plate case |
96-Well Deep-Well Plate |
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Hampton |
HR3-081 |
72 Well Microbatch Plate, Greiner 654102 |
untreated, hydrophobic - 270 plate case |
72-Well Under-Oil Crystallization Plate |
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Hampton |
HR3-125 |
Swissci 3 Well Midi Crystallization Plate in UVP (40) |
40 plate case |
3-Microdrop/96-Well Hanging Drop Plate |
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Hampton |
HR3-158 |
Cryschem Plate |
24 plate case |
24-Well Hanging Drop Plate |
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Hampton |
HR3-231 |
22 mm x 0.22 mm Siliconized circle cover slides |
1.0 ounce pack (~120 slides) |
Circular Silanized Glass Coverslips |
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Hampton |
HR3-233 |
22 mm x 0.22 mm Siliconized circle cover slides |
10.0 ounce case (~1,200 slides) |
Circular Silanized Glass Coverslips |
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Hampton |
HR3-609 |
Crystal Clear Sealing Film |
100 pack |
Crystallization Plate Sealing Film |
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Hampton |
HR4-216 |
Crystal Crusher |
5 pack |
Crystal Crusher |
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Hampton |
HR4-217 |
Crystal Probe |
12 pack |
Disposable Stainless Steel Crystal Needles |
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Hampton |
HR4-733 |
CrystalCap |
with Vial - 60 pack |
Crystal Loop Base |
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Hampton |
HR4-811 |
Micro-Tools Set |
each |
Crystal Handling Tool Set, including 8 crystal probes of different shapes |
In addition to the aforementioned products, XMJ also provides various specifications of crystallization plates, loops, microbridges, sealing compounds, pucks, and other tool consumables, as well as complete toolkits. These products help researchers conduct experiments faster and more efficiently. For inquiries and orders, please feel free to send email to info@xmjsci.com
As the official authorized distributor of Hampton Research in China, XMJ is committed to providing users with excellent technical support and thoughtful after-sales service. XMJ has always adhered to its core philosophy of "We promise what we can do. We do what we have promised" to deliver high-quality services to users. If you want to learn more about product information, please c feel free to send email to info@xmjsci.com or visit the official website at guanghecar.com for inquiries and more details.
References and Readings
1.Der Chemismus in der tierescher Organization, Hunefeld, F 1. (1840)p.160.Leipzig University, Germany.
2.A brief h istory of protein crystal growth, MePherson, A. (1991). J. Cryst.Growth, 110,1-10.
3 über die Eiweisskorper verschiedenen Oelsamen, Ritthausen, H. (1880)Pfluegers Arch. 21,81-104.
4 The proteins of the Brazil nut, Osborne T.(1891). Am. Chem. J. 13,212-218.
5 Crystalline insulin., Abel, 」.J., Geiling, E. M. K., Roultier, O. A., Bell, F. M.&Wintersteiner, 0.(1927).]. Pharmacol. Exp. Ther. 31,65-85.
6 The Enzymes, Sumner, J.B.& Somers, G.F(1943). New York: Aca-demic Press.
7 Crystalline enzymes, Northrop, M., Kunitz, M. & Herriott, R. M. (1948)New York: Columbia University Press.
8 Present at the flood: How structural molecular biology came about,Dickerson,R.E.(2005).FASEB J.20,809-810.
9 Preparation and Analysis of Protein Crystals, McPherson, A.(1982)New York: lohn Wiley & Sons.
10.Current approaches to macromolecular crystallization. McPherson, A. (1990).Eur.Biochem.189,1-23.
11.Crystallization of Biological Macromolecules, McPherson, A.(1999)Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
12Introduction to protein crystallization, McPherson, A. and Gavira, J.A.(2014) Acta Crystallographica F, Volume 70, Part 1, 2-20.
13.Some Words of Advice from an Old Hand, Alexander McPherson, pages1-9, in Protein Crystallization, Second Edition, Edited by Terese Berg-fors. International University Line, 2009.
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